Inhibitory effect of grapefruit seed extract (GSE) on avian pathogens.

The inhibitory activities of grapefruit seed extract (GSE) on avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis (SI) and Escherichia coli (EC) were evaluated. Original GSE contained 0.24% benzalkonium chloride (BZC), however, 0.0025% BZC solution could not inactivate bacteria. The activity of diluted GSE (×100, ×500 and ×1,000 with redistilled water) against selected viruses and bacteria was evaluated in this study. The GSE solutions were incubated with the pathogens over a period of time after which the remaining viruses were titrated and the bacterial colonies were counted. In the presence of organic material-5% fetal bovine serum (FBS), the test solutions were sprayed at 1 cm and 30 cm distances to test the efficacy of GSE in a spray form. Furthermore, the efficacy of GSE against bacteria on clothes was tested using non-woven cloth. GSE×100 reduced the viral titer of both AIV and NDV even in 5% FBS condition. IBDV showed high resistance to GSE. GSE×1,000 inactivated both SI and EC within 5 sec, even in the presence of 5% FBS. The disinfectant was able to maintain its efficacy in the spray form at 30 cm distance. GSE was also effective against SI and EC inoculated on fabric. GSE is a potential novel disinfectant against viruses and bacteria, effective even within a short contact time.

Bactericidal efficacy of a quaternary ammonium compound with food additive grade calcium hydroxide toward Salmonella Infantis and Escherichia coli on abiotic carriers.

The bactericidal efficacies of 0.2% food additive grade calcium hydroxide (FdCa(OH)2) solution, a quaternary ammonium compound (QAC) diluted at 1:500 (QACx500) and their mixture-Mix500 (FdCa(OH)2 powder added at final concentration 0.2% to QACx500)-were investigated at two different temperatures (room temperature (RT) (25 ± 2°C) and 2°C), using varying contact time, with or without presence of organic materials (5% fetal bovine serum: FBS), either in suspension or on abiotic carrier (steel, rubber and plastic). In the suspension test, QACx500 could inactivate Salmonella Infantis at effective level (≥3 log reductions), within 30 sec and 5 sec, respectively, with or without 5% FBS at RT; however, at 2°C it required 30 min and 1 min, respectively. Mix500 revealed the same efficacy as QACx500 at RT, but, at 2°C it required 1 min and 30 sec, respectively with or without FBS. Whereas, 0.2% FdCa(OH)2 solution alone could inactivate S. Infantis within 1 min and 3 min, respectively at RT and 2°C, even with 5% FBS. In the carrier test, single disinfectant required bit more (3 or 5 min) contact time to reduce bacterial load (S. Infantis or Escherichia coli) down to the effective level on rubber surface than that on steel and plastic surface. However, Mix500 could inactivate both bacteria on carrier surfaces within 1 min, even at 2°C. Thus, synergistic effects were observed in the suspension test and the carrier test at both temperatures toward both bacteria.

Bactericidal Efficacy of Food Additive-Grade Calcium Hydroxide Against Salmonella Infantis on Eggshells.

The bactericidal efficacy of food additive-grade calcium hydroxide [FdCa(OH)2] was evaluated for inactivation of Salmonella Infantis and Salmonella Enteritidis in liquid and Salmonella Infantis on contaminated eggshells. The activity of FdCa(OH)2 was also compared with that of sodium hypochlorite (NaOCl) containing 150 ppm chlorine (150 ppm NaOCl). FdCa(OH)2 solutions (0.1% and 0.2%) in the presence or absence of organic materials (5% calf serum [CS]) at pH 12.6 were used to inactivate Salmonella Infantis and Salmonella Enteritidis in a reaction tube or on eggshells artificially contaminated with Salmonella Infantis. Both 0.1% and 0.2% FdCa(OH)2 were capable of inactivating Salmonella Infantis and Salmonella Enteritidis in liquid at >3 log10 colony-forming units (CFU)/ml within 3 and 1 min of contact time, respectively, even in the presence of 5% CS. Additionally, 0.1% and 0.2% FdCa(OH)2 reduced bacterial levels on contaminated eggshells to >3 log10 CFU/ml, within 3 and 1 min, respectively, in the presence of 5% CS. Without CS, 0.1% and 0.2% FdCa(OH)2 could reduce bacteria on eggshells to >3 log10 CFU/ml within 1 min and 30 sec, respectively. In contrast, 150 ppm NaOCl solution could not inactivate bacteria on eggshells down to >3 log10 CFU/ml within 3 min contact time, either with or without CS, and no bacterial reduction was observed in redistilled water. The findings of the present study indicate that FdCa(OH)2 solution has high efficacy against foodborne bacteria and may be a good candidate for enhancement of biosecurity at farms and egg processing plants.

Virucidal efficacy of food additive grade calcium hydroxide against surrogate of human norovirus.

An alkaline agent, namely, food additive grade calcium hydroxide (FdCa(OH)2) in the solution, powder and suspension forms was evaluated as a virucidal agent, using a murine norovirus (MNV) as the surrogate for human norovirus. The main constituent of FdCa(OH)2 is Ca(OH)2, which has pH 13 in 0.17% solution. The results showed that 0.17% FdCa(OH)2 solution could inactivate MNV within 30s even in the presence of organic materials (5% fetal bovine serum (FBS)). In a contaminated surface experiment, MNV with 5% FBS was inoculated on rayon sheets, and the result showed FdCa(OH)2 solution could markedly reduce virus titer within 1min. When mouse feces were spiked with MNV and FdCa(OH)2 powder as 10% and 20% w/w was added to the feces, these concentrations could inactivate the virus within 30min and 15min, respectively. Whereas, FdCa(OH)2 suspension at 2.5% and 5% could inactivate the virus within 30min and at 1% within 45min. These and additional results obtained here indicate that FdCa(OH)2 is an effective virucidal agent against MNV, and can serve as a useful alternative disinfectant for inactivation and prevention of human norovirus in house and hospital.

Virucidal Efficacy of a Quaternary Ammonium Compound with Food Additive-Grade Calcium Hydroxide Toward Avian Influenza Virus and Newcastle Disease Virus on Abiotic Carriers.

The virucidal efficacies of a 0.2% food additive-grade calcium hydroxide [FdCa(OH)2] solution, a quaternary ammonium compound (QAC) diluted at 1:500 (QACx500), and their mixture [Mix500; FdCa(OH)2 powder added at a final concentration of 0.2% to QACx500] were investigated as fomites for avian influenza virus (AIV) and Newcastle disease virus (NDV) on abiotic carriers (steel, rubber, and plastic) at two different temperatures (room temperature [RT; 25 ± 2 C] and 2 C). These viruses were seeded on coupons (5 cm×5 cm) of rubber, steel, or plastic with 5% fetal bovine serum. After complete drying, the coupons were covered with the test solutions at RT or 2 C. After fixed incubation periods, viruses were recovered from the coupons and titrated. At RT, Mix500 required a short time (3 min) to inactivate AIV and NDV to effective levels (≥3 log virus reduction) on rubber, steel, and plastic carriers compared with QAC or FdCa(OH)2. At low temperature, QACx500 inactivated AIV on steel and plastic carriers to effective levels within 60 min, whereas Mix500 did so within 10 min. QACx500 and FdCa(OH)2 solutions could inactivate NDV on steel and plastic carriers within 20 and 10 min, respectively, and Mix500 could do so within 3 min. Viruses on the carriers required longer incubation periods for inactivation at 2 C than at 25 C. These results demonstrate desirable synergistic virucidal effects of Mix500 for important poultry viruses on abiotic carriers, while indicating high applicability within poultry farming.

Eficacia virucida de un compuesto de amonio cuaternario con hidróxido de calcio con grado aditivo alimentario contra el virus de la influenza aviar y el virus de la enfermedad de Newcastle en vehículo abióticos. Las eficacias virucidas de una solución de hidróxido de calcio [FdCa(OH)2] con grado aditivo alimentario al 0.2%, de un compuesto de amonio cuaternario (QAC) diluido a 1:500 (QACx500) y su mezcla [Mix500; FdCa(OH)2 en polvo agregado en una concentración final de 0.2% en QACx500] se investigaron en forma de fómites para el virus de la influenza aviar (AIV) y para el virus de la enfermedad de Newcastle (NDV) en vehículos abióticos (acero, hule y plástico) a dos temperaturas diferentes (temperatura ambiente [RT; 25 ± 2 C] y 2 C). Estos virus se sembraron en placas (5 cm x 5 cm) de hule, acero o plástico con suero bovino fetal al 5%. Después del secado completo, los cupones se cubrieron con las soluciones bajo ensayo a temperatura ambiente o 2 C. Después de los períodos de incubación fijos, los virus se recuperaron de las placas y se titularon. A temperatura ambiente, la mezcla Mix500 requirió un corto tiempo (tres minutos) para desactivar a los virus de influenza y de Newcastle a niveles efectivos (reducción igual o mayor de tres logaritmos en el título viral) en las placa de hule, acero y plástico en comparación con el tratamiento QAC o FdCa(OH)2. A baja temperatura, el tratamiento QACx500 inactivó al virus de la influenza en placas de acero y plástico a niveles efectivos dentro de 60 minutos, mientras que el tratamiento Mix500 lo hizo en 10 minutos. Las soluciones QACx500 y FdCa(OH)2 pudieron inactivar al virus de Newcastle en las placas de acero y plástico dentro de 20 y 10 minutos, respectivamente, y el tratamiento Mix500 pudo hacerlo dentro de tres minutos. Los virus en los vehículos requirieron períodos de incubación más prolongados para la inactivación a 2 C que a 25 C. Estos resultados demuestran los efectos virucidas sinérgicos deseables del tratamiento Mix500 para virus aviares importantes en vehículos abióticos, además de que indican una alta aplicabilidad dentro de la avicultura.

Enhancement of bactericidal effects of sodium hypochlorite in chiller water with food additive grade calcium hydroxide.

An alkaline agent, namely food additive grade calcium hydroxide (FdCa(OH)2) in solution at 0.17%, was evaluated for its bactericidal efficacies in chiller water with sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine. Without organic material presence, NaOCl could inactivate Salmonella Infantis and Escherichia coli within 5 sec, but in the presence of fetal bovine serum (FBS) at 0.5%, the bactericidal effects of NaOCl were diminished completely. FdCa(OH)2 solution required 3 min to inactivate bacteria with or without 5% FBS. When NaOCl and FdCa(OH)2 were mixed at the final concentration of 200 ppm and 0.17%, respectively, the mixed solution could inactivate bacteria at acceptable level (103 reduction of bacterial titer) within 30 sec in the presence of 0.5% FBS. The mixed solution also inhibited cross-contamination with S. Infantis or E. coli on chicken meats. It was confirmed and elucidated that FdCa(OH)2 has a synergistic effect together with NaOCl for inactivating microorganisms.

Durability of alkaline agents' bactericidal efficacies in litter under field conditions.

Alkaline agents are well-known for their disinfection capacities against pathogens even at the presence of organic materials, but the durability of their bactericidal efficacies under field conditions is unknown. Therefore, within the present study, two alkaline agents, namely bioceramic (BCX) derived from chicken feces and food additive grade Ca(OH)2 (FdCa(OH)2) derived from natural lime stone, were evaluated for the persistence of their bactericidal efficacies in litter, under simulated field conditions. BCX powder mixed at 50% concentration in litter or FdCa(OH)2 powder at 20% concentration in litter lost their bactericidal efficacies at 3 days post exposure of chicks, and thereafter, both mentioned alkaline agents could not inactivate bacteria down to the acceptable level (≥3 log10 CFU/ml reduction).

Accuracy of the evaluation method for alkaline agents' bactericidal efficacies in solid, and the required time of bacterial inactivation.

An alkaline agent, namely food additive grade calcium hydroxide (FdCa (OH)2) in the powder form, was evaluated for its bactericidal efficacies in chicken feces at pH 13. The point for this evaluation was neutralization of the alkaline agent's pH at the time of bacterial recovery, since otherwise the results are substantially misleading. Without neutralization of the FdCa (OH)2 pH, the spiked bacteria were killed within min at the time of recovery in aqueous phase, but not in the solid form in feces, hence, it has been demonstrated that when bacteria were in solid, it took longer time than in liquid for the alkaline agent to inactivate them down to the acceptable level (≥3 log10 CFU/ml).

Virucidal Properties of Bioceramic Derived from Chicken Feces pH 13 and its Stability in Harsh Environments.

Bioceramic derived from chicken feces (BCX) is a material produced by a sintering process for the purpose of use in animal farms to control livestock infectious diseases. In the present study, BCX at pH 13 was evaluated for the durability of its virucidal activity in simulated field conditions. First it was shown that BCX had activity toward Newcastle disease virus, infectious bursal disease virus, and goose parvovirus within 3 min and toward avian influenza virus (AIV) within 1 hr. BCX was further tested by keeping it under simulated harsh environmental conditions with sunlight for several weeks as well as by repeatedly soaking it with water and drying under sunlight many times. After sampling every 2 consecutive weeks and every 2 (of 9) consecutive resuspensions, BCX was evaluated for its efficacy against AIV. Evaluation under the harsh conditions illustrated that BCX could retain its satisfactory efficacy toward AIV throughout 7 wk and through 9 resuspensions. It is hence concluded that BCX is an excellent material for applying in livestock farming as a trapping disinfectant, due to its efficacy to inactivate various viruses, and that this efficacy is prolonged even under harsh environmental conditions.

TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus.

Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV) in vivo. IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo.

Inactivation of bacteria on surfaces by sprayed slightly acidic hypochlorous acid water: in vitro experiments.

The capacity of slightly acidic hypochlorous acid water (SAHW), in both liquid and spray form, to inactivate bacteria was evaluated as a potential candidate for biosecurity enhancement in poultry production. SAHW (containing 50 or 100 ppm chlorine, pH 6) was able to inactivate Escherichia coli and Salmonella Infantis in liquid to below detectable levels (≤2.6 log10 CFU/ml) within 5 sec of exposure. In addition, SAHW antibacterial capacity was evaluated by spraying it using a nebulizer into a box containing these bacteria, which were present on the surfaces of glass plates and rayon sheets. SAHW was able to inactivate both bacterial species on the glass plates (dry condition) and rayon sheets within 5 min spraying and 5 min contact times, with the exception of 50 ppm SAHW on the rayon sheets. Furthermore, a corrosivity test determined that SAHW does not corrode metallic objects, even at the longest exposure times (83 days). Our findings demonstrate that SAHW is a good candidate for biosecurity enhancement in the poultry industry. Spraying it on the surfaces of objects, eggshells, egg incubators and transport cages could reduce the chances of contamination and disease transmission. These results augment previous findings demonstrating the competence of SAHW as an anti-viral disinfectant.

Calcinated egg shell as a candidate of biosecurity enhancement material.

Calcinated egg shell (Egg-CaO), of which the main component is calcium oxide, was evaluated in the forms of powder and aqueous solutions for their efficacies as disinfectants against avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis and Escherichia coli. Egg-CaO powder inactivated these viruses within 3 min in the presence of 33% of fetal bovine serum (FBS). In Egg-CaO solutions, except AIV, all pathogens were inactivated within 1 hr, even in the presence of 5% of FBS. Without FBS, all pathogens, except AIV, were inactivated within 3 min, and AIV within 1 hr. In addition, persistence of virucidal activity against AIV and NDV of Egg-CaO powder was confirmed after exposure to sunlight for 2 weeks or resuspension with water for 7 times, simulating field harsh environments. Chick growth test was conducted to ensure the safety of the use of Egg-CaO powder in chicken cages and showed that it is safe to add Egg-CaO in litter or feed. In conclusion, Egg-CaO can be useful for the enhancement of biosecurity at farms.